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Transcript
A M E R S H A M B I O S C I E N C E S Phosphoamino Acid Analysis by Thin Layer Electrophoresis Contributed by Charles W. Mahoney, Noriyuki Nakanishi, Motoaki Ohashi Tanabe Pharmaceutical Company Ltd., Toda, Japan Application Note #2 Multiphor® II A) Ninhydrin B) Autoradiograph Keywords: Phosphoprotein; p53 Introduction Proteins are often phosphorylated in vivo on Ser, Thr, and/or Tyr residues. As this phosphorylation often serves a regulatory role in protein and cellular function, identification of the phosphorylated amino acid(s) in a particular protein is important in signal transduction research. This is commonly done by hydrolyzing the 32P-labeled phosphoprotein and separating the hydrolysate by thin layer electrophoresis on cellulose TLC plates. The phosphorylated amino acids are identified by comparing the mobility of phosphoamino acids visualized by autoradiography to the mobility of phosphoamino acid standards visualized with ninhydrin [1-6]. P-Ser P-Thr P-Tyr BPB Here we describe a protocol for rapid, reproducible phosphoamino acid analysis by semi-dry thin layer electrophoresis in the Multiphor II electrophoresis unit [5]. O rigin Results This technique was applied in order to identify the phosphorylated amino acid(s) in immunoprecipitated p53 that had been labeled with 32P phosphate in vivo. The results clearly indicate the presence of phosphoserine alone (Figure 1). This technique has proved to be rapid and reproducible, and it is performed on a widely available flatbed electrophoresis apparatus. Fig. 1. Phosphoamino acid analysis of 32P-labeled p53 by semi-dry cellulose thin-layer electrophoresis. A) Photograph of the TLC plate stained with ninhydrin. B) Autoradiograph of the TLC plate. Positions of phosphoserine, phosphothreonine, phosphotyrosine and the bromophenol blue marker dye are as indicated. Figure used with permission of Academic Press [5]. Methods SDS-PAGE and electrotransfer A sample containing 32P-labeled protein was separated by standard Laemmli SDS-PAGE and blotted onto a polyvinylidine difluoride (PVDF) membrane (Immobilon P, Millipore). The SDS-PAGE can be done using a SE 600 or SE 400 gel electrophoresis unit. The electrotransfer can be done in a semi-dry mode on a TE 70 or TE 77 SemiPhor™ semi-dry transfer unit, or in tank mode in a TE 62 or TE 42 Transphor™ tank transfer unit. The equipment manufacturer’s instructions should be followed for the SDS-PAGE and electrotransfer. 80-6413-07 Rev A / 11-97 Semi-dry cellulose thin layer electrophoresis Sample preparation Solutions 6 N HCl (6 N Hydrochloric acid, 10 ml) Concentrated HCl (~12 N) Distilled water Materials Glass-backed cellulose TLC plates, 20 x 20 cm, or 10 x 20 cm are available from Merck (Germany), catalog number 1.05716.0000. These plates are distributed in North America by: 5 ml 5 ml 30% methanol, 0.1 N HCl (30% methanol, 0.1 N Hydrochloric acid, 10 ml) Methanol Concentrated HCl (~12 N) Distilled water EM Separations Technology 480 Democrat Rd. PO Box 70 Gibbstown, NJ 08027 Tel: 609-224-0742 Fax: 609-423-4389 3 ml 83 µl to10 ml Phosphoamino Acid Standards (0.5% phosphoserine, 0.5% phosphothreonine, 0.5% phosphotyrosine, 1 ml) O-Phospho-L-serine (FW 185.1) O-Phospho-L-threonine (FW 199.1) O-Phospho-L-tyrosine (FW 261.2) Distilled water For a list of other international distributors, contact: Merck KGaA 64271 Darmstadt Frankfurter Straße 250 Tel: 49-6151-72-0 Fax: 49-6151-72-2000 5 mg 5 mg 5 mg 1 ml Solutions Bromophenol Blue Tracking Dye (0.1% bromophenol blue, 1 ml) Bromophenol blue (FW 691.9) Distilled water Electrophoresis Buffer (5% (v/v) acetic acid, 0.5% (v/v) pyridine, pH 3.5, 100 ml) 1 mg 1 ml Acetic acid (glacial) Pyridine Distilled water Following transfer of the 32P-labeled proteins to the PVDF membrane, the membrane was marked with luminescent or radioactive markers for orientation, covered with plastic wrap and exposed to x-ray film with the protein side towards the film (the length of the exposure will vary with the abundance of the protein and its phosphate content). After developing the autoradiograph, the membrane was aligned with the x-ray film on a light box. A razor blade was used to excise the area of PVDF membrane corresponding to the 32 P-labeled protein band of interest. 5 ml 500 µl 94.5 ml The MultiTemp® III thermostatic circulator was connected to the Multiphor II electrophoresis unit and pre-cooled to 16 °C. The glass backed cellulose TLC plates were marked with a blunt soft pencil at 3 and 9 cm for the origin and migration point for the bromophenol blue tracking dye, respectively. Phosphoamino Acid Standards (1 µ1, ~25 nmol of each phosphoamino acid) were applied to each lane at the origin using a glass capillary or 1 µl pipetter, drying between each application with a cool hair dryer. Hydrolyzed 32P-labeled protein was applied in a similar manner to each spot at the same origin pre-spotted with Phosphoamino Acid Standards. Bromophenol Tracking Dye (1 µl) was applied to a separate lane. The TLC plate was gently sprayed with Electrophoresis Buffer and placed in the center of and parallel to the long axis of the Multiphor II cooling plate with the origin towards the cathodic (-) end. Amersham Biosciences CleanGel™ electrode strips were cut to the width of the TLC plate (5 x 10.5 cm or 5 x 20.5 cm) and pre-soaked with Electrophoresis Buffer The immobilized 32P-labeled protein was hydrolyzed by incubating the membrane slice in 200 µl 6 N HCl for 2 h at 110 °C in a securely capped 1.5 ml polypropylene microcentrifuge tube. The amino acids were eluted from the membrane with 30% methanol, 0.1 HCl (3 changes, 200 µl each). The 6 N HCl hydrolysate and the three eluates were pooled and dried in a speed-vac, or by lyophilization. The sample was resuspended in 10 µl of distilled water. 2 Glass Plates Electrode Plate Detection Cathod Solutions Ninhydrin Solution (0.2% (w/v) ninhydrin in ethanol, 10 ml) Ninhydrin 20 mg Ethanol (reagent grade) 10 ml Anode TLC Plate The TLC plate was removed from the Multiphor II unit and dried for 10 min with a hot hair drier. The plate was sprayed with Ninhydrin Solution and blown with a hot hair drier for 10 min to visualize the phosphoamino acid standards. The TLC plate was exposed to X-ray film to determine the position of the 32P phosphoamino acids. Spotting radioactive or luminescent markers onto the dried TLC plate helped in aligning the autoradiogram to the TLC plate. CleanGel Electrode Strip Fig. 2. Schematic of the Multiphor II flatbed electrophoresis unit set up for phosphoamino acid analysis. (See text for details). Redrawn from [5]. (10 ml or 20 ml depending, respectively, on the length of the CleanGel electrode strips). The wet strips were overlapped 2 cm onto each end of the TLC plate and three glass plates the same size and thickness of the TLC plate were placed on top of the TLC plate with the wicks in place. TLC plates from which the cellulose layer has been scraped off can be used. (The weight of the glass plates was found to be critical for maintaining good electrode strip to TLC plate contact and the proper humidity). The interelectrode distance was set to 24 cm and the electrode plate was placed over the TLC plate, glass plates and buffer-soaked electrode strips so that the electrodes rested on the electrode strips with the cathode towards the origin (see Figure 2). The lid was placed on the Multiphor II and connected to a high voltage power supply set for 1000 V, with current and power limits set at 30 mA and 30 W, respectively. Electrophoresis was run at constant voltage for 40-50 min and terminated when the Bromophenol Blue Tracking Dye had migrated 6 cm. Note in Proof It has been found that use of the following alternative electrophoresis buffer results in greater separation among the three phosphoamino acids (data not shown) [6]. Electrophoresis Buffer (5.93% (v/v) acetic acid, 0.73% (v/v) formic acid, 0.33% (v/v) pyridine, 0.33 mM EDTA, pH 2.5, 100 ml) Acetic acid (glacial) Formic acid (88%) Pyridine 0.5 M EDTA, disodium salt Distilled water 3 5.93 ml 830 µl 330 µl 66 µl 92.8 ml References 1. Cooper, J.A, Sefton, B.M. and Hunter, T. Detection and quantification of phosphotyrosine in proteins. Meth. Enzymol. 99, 387-402 (1983). 2. Kamps, M.P. and Sefton, B.M. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal. Biochem. 176, 22-27 (1989). 3. van der Geer, P., Luo, K., Sefton, B.M. and Hunter, T. Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates. Protein Phosphorylation - A Practical Approach. Oxford University Press Inc., NY (1993). 4. van der Geer, P and Hunter, T. Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates. Electrophoresis 15, 544-554 (1994). 5. Mahoney, C.W., Nakanishi, N. and Ohashi, M. Phosphoamino acid analysis by semidry electrophoresis on cellulose thin-layer plates using the Amersham Biosciences or Atto flatbed apparatus. Anal. Biochem. 238, 96-98 (1996). 6. Jelinek, T. and Weber, M.J. Optimization of the resolution of phospho-amino acids by one-dimensional thin-layer electrophoresis. BioTechniques 15,629-630 (1993). Ordering Information Code No. 80-6171-58 80-6154-86 80-6210-34 80-6211-86 80-6205-98 80-6209-58 18-1018-06 18-1102-77 18-1102-78 19-3500-00 18-1035-33 17-1324-01 17-1329-01 Item Description SE 600 Dual Vertical Gel Unit SE 400 Sturdier Vertical Gel Unit TE 70 SemiPhor Semi-Dry Transfer Unit TE 77 SemiPhor Semi-Dry Transfer Unit TE 42 Transphor Tank Transfer Unit TE 62 Transphor II Tank Transfer Unit Multiphor II Electrophoresis Unit MultiTemp® III Refrigerated Bath Circulator, 100-120 V MultiTemp III Refrigerated Bath Circulator, 200-220 V EPS 3500 Power Supply CleanGel electrode strips, package of 12 EDTA, disodium salt, 100 g Bromophenol Blue, 10 g